Luminescence microscopy is a classic application of light microscopy for studying biological preparations. To this end, certain dyes (known as phosphores or fluorophores) are used to specifically mark specimens, e.g., cell components. As mentioned [sic], the specimen is illuminated with illuminating radiation that produces excitation radiation [sic] and the thereby excited luminescence radiation is detected with suitable detectors. Normally, the microscope for this purpose comprises a dichroic beam splitter in combination with block filters that separate the luminescence radiation from the excitation radiation and thereby make it possible to observe [the beams] separately. This method makes it possible to visualize individual cell components dyed in different colors under the microscope. It is, of course, also possible for several components of a preparation to be simultaneously dyed with different dyes which accumulate specifically in different structures of the preparation. This method is known as multi-luminescence. It is also possible to examine specimens that luminesce on their own, i.e., without the addition of dyes.
As generally accepted, luminescence in this context is used as a broader term for phosphorescence and fluorescence, i.e., it covers both phenomena. Thus, when this document makes reference to fluorescence, this should be understood as pars pro toto rather than as a restriction.
It is also known that, to investigate specimens, laser scanning microscopes (abbreviated as LSM) can be used, which, by means of a confocal detection configuration (known as a confocal LSM) or nonlinear specimen interaction (so-called multiphoton microscopy), display, out of a fully three-dimensionally illuminated image, only the plane that is located in the focal plane of the objective lens. Thus, an optical section is obtained, and the recording of a plurality of optical sections in different depths of the specimen subsequently makes it possible to generate, by means of a suitable data processing system, a three-dimensional image of the specimen, which image is composed of the various optical sections. Thus, laser scanning microscopy is useful when thick preparations are to be studied.
Obviously, a combination of luminescence microscopy and laser scanning microscopy can be used as well; in this case, a luminescent specimen is imaged in planes of various depths by means of an LSM.
U.S. Pat. No. 5,043,570 describes an attempt to enhance the resolution by means of “oversampling.”
This does not lead to a markedly improved resolution below the diffraction limit of the microscope.
Due to the laws of physics, the optical resolution of a light microscope, including that of an LSM, is invariably diffraction-limited. For optimum resolution within these limits, special illumination configurations are known, such as the 4Pi configuration or configurations with standing-wave fields. As a result, the resolution, in particular in an axial direction, can be markedly improved over that of a conventional LSM. In addition, using nonlinear depopulation methods, the resolution can be enhanced to a factor of up to 10 compared to a diffraction-limited confocal LSM. Such a method has been described, for example, in U.S. Pat. No. 5,866,911. As to depopulation methods, several different approaches are known, such those described, for example, in DE 44 16 558 C2, U.S. Pat. No. 6,633,432 or DE 103 25 460 A1.
U.S. Pat. No. 5,867,604 discusses another high-resolution microscopy method in which an object with a periodic structure is scanned.
A similar method for enhancing the resolution is discussed in EP 1 157 297 B1. This method is said to utilize non-linear processes by means of structured illumination. This document mentions the saturation of fluorescence as an example of non-linearity. The method described claims to achieve a shift of the object space spectrum relative to the transfer function of the optical system by means of structured illumination. In concrete terms, a shift of the spectrum means that object space frequencies V0 are transferred at a space frequency of V0-Vm, with Vm being the frequency of the structured illumination. At a given maximum space frequency that the system can transfer, this enables the transfer of space frequencies of the object that are above the maximum frequency of the transfer function by shifting frequency Vm. This approach requires a reconstruction algorithm for image generation and the evaluation of several image acquisitions for one image. Another disadvantage of this method is that the specimen, in areas outside the detected focus, is unnecessarily exposed to radiation since the necessary structured illumination passes through the entire specimen volume. Furthermore, this method can currently not be used for thick specimens, because out-of-focus excited fluorescence also reaches the detector as a background signal and thus dramatically reduces the dynamic range of the detected radiation.
A method which, irrespective of laser scanning microscopy, achieves a resolution beyond the diffraction limit is known from WO 2006/127692 and DE 10 2006 021 317. This method known by the acronym PALM (Photo Activated Light [sic; Localization] Microscopy) uses a marker substance which can be activated by means of an optical activation signal. Only when the marker substance is in the activated state is it possible for the substance to be excited with excitation radiation to induce a certain fluorescence radiation. Even when exposed to excitation radiation, non-activated molecules of the marker substance do not emit any fluorescence radiation, or at least do not emit noticeable fluorescence radiation. Thus, the activating radiation switches the marker substance into a state in which it can be excited to fluoresce. Other types of activation, e.g., thermal-type activation, are possible as well. Therefore, one generally speaks of a switching signal. In the PALM method, the switching signal is applied in such a manner that that at least a certain portion of the activated marker molecules are at a distance from the neighboring activated molecules so that, as measured against the optical resolution of microscopy, they are separate or can be subsequently separated. This means that the activated molecules are at least to a large extent isolated. After absorption of the luminescence radiation, the center of the radiation distribution associated with the resolution limit of these isolated molecules is determined for these molecules and, based thereon, the location of the molecules is mathematically determined with higher accuracy than optical imaging actually allows. In the English-language literature, this enhanced resolution obtained by mathematically determining the center of the diffraction distribution is also referred to as “superresolution.” It requires that at least some of the activated marker molecules, with the optical resolution with which the luminescence radiation is detected, are distinguishable, i.e., isolated, in the specimen. If this is the case, such molecules can be localized with enhanced resolution.
To isolate individual marker molecules, the PALM method relies on the fact that the probability with which a marker molecule is activated by the activation radiation after receiving the switching signal of a given intensity, e.g., a photon, is the same for all molecules. Thus, by way of the intensity of the switching signal and thus the number of photons that impinge upon a unit area of the specimen, it can be ensured that the probability that marker molecules that are present in a given area of the specimen are activated is so low that there are sufficient areas in which only distinguishable marker molecules emit fluorescence radiation within the optical resolution. By properly choosing the intensity, e.g., the photon density, of the switching signal, it can be ensured that marker molecules in locations that are preferably isolated only relative to the optical resolution are activated and subsequently emit fluorescence radiation. Subsequently, the center of the diffraction-associated intensity distribution is mathematically determined for these isolated molecules and thus the location of the marker molecule with enhanced resolution is determined. To image the entire specimen, isolation of the marker molecules of the subset by introduction of activation radiation, subsequent excitation and fluorescence radiation imaging is repeated until preferably all marker molecules were at one time included in a subset and isolated within the resolution of the image.
The PALM method has the advantage that neither activation nor excitation requires a high spatial resolution. Instead, both activation and excitation can take place in wide-field illumination.
As a result, the marker molecules are statistically activated in subsets by properly choosing the intensity of the activation radiation. To generate an overall image of a specimen in which the locations of all marker molecules can be mathematically determined with a resolution that is, e.g., beyond the diffraction limit, it is therefore necessary for a plurality of individual images to be evaluated. This may involve up to 10,000 individual images. This means that large amounts of data are processed and, accordingly, the measurement may take a long time. Even just capturing an overall image requires several minutes, which is essentially determined by the readout rate of the camera used. The location of the molecules in the individual images is determined by means of complicated mathematical procedures, such as have been described, for example, by Egner et al., Biophysical Journal, pp. 3285-3290, Volume 93, November 2007. Typically, it takes 1-2 h to process all individual images and to assemble them to create a high-resolution overall image, i.e., one image, in which the locations of the marker molecules with a resolution beyond the diffraction limit are identified.
Other articles on high-resolution methods include:
Hell, S. W. (2007): “Far-Field Optical Nanoscopy,” Science 316, pp. 1153-1158,
and on SAX (Saturated Excitation) Microscopy: Fujita et al., Phys. Rev. Lett. (2007), Yamanaka et al., J. Biomed. Opt. (2008).
The prior-art high-resolution methods have a number of disadvantages:
The disadvantage of the STED method is the availability of dyes and the high laser intensity required. The RESOLFT/GSD method requires a high number of switching cycles. In the PALM/STORM method, the image generation rate is slow, and SAX microscopy causes considerable bleaching of the dyes.